Abstract

The progression of colorectal cancer (CRC) from normal colonic epithelium to the malignant phenotype is associated with diverse genetic and epigenetic changes, including mutations of APC or beta-catenin genes. Extensive experimental and epidemiological evidence also suggest that differences in diet contribute to more variation in CRC incidence than any other factor. For example, several animal experimental studies support the notion that dietary Ca2+ prevents the development of CRC. The precise mechanism(s) mediating the chemopreventive properties of Ca2+ remain incompletely understood. The extracellular Ca2+-sensing receptor (CaR), a GPCRs originally cloned from parathyroid chief cells, plays a major role in the control of extracellular Ca2+ concentration by regulating parathyroid hormone secretion. However, subsequent studies demonstrated that the CaR is also expressed in other tissues and organs, including the entire gastrointestinal (GI) tract. Interestingly, CaR expression in colonic carcinomas is greatly reduced or lost, suggesting that the suppression of its expression may be associated with abnormal differentiation and malignant progression. Here, we assessed the role of the CaR in colonic epithelial cells proliferation. In order to elucidate the mechanisms involved, we examined in vitro changes in beta-catenin phosphorylation and intracellular distribution and cellular proliferation triggered by CaR stimulation. We found that CaR stimulation promotes, in human epithelial cells derived from normal colon and from colorectal adenocarcinoma, a decrease of up to 60% in beta-catenin Ser-552 phosphorylation, a residue involved in beta-catenin nuclear import and transcriptional activity regulation. This change in phosphorylation was accompanied by a redistribution of beta-catenin from the cytoplasm and nuclei to the plasma membrane and by a marked proliferation inhibition. To determine the role of the CaR in the proliferation of colonic cells in vivo, we employed a novel mouse model where the expression of the CaR was specifically knocked out in the GI tract. The results show that the crypts in the distal colon are deeper in knock out (KO) mice (389 ± 7.39 Arbitrary Units, mean ± SEM) than controls littermates (268 ± 6.35 AU) P < 0.0001. Similarly, the crypts in the proximal colon are deeper in KO mice (289 ± 7.02 AU) than controls (232 ± 5.36 AU) P < 0.0001. We also found significant morphological differences in the crypt’ structure of several regions in the proximal colon of CaR KO mice, including the absence of a well-organized surface layer of epithelial cells and the presence of disorganized crypts and numerous enlarged globet cells. Further analysis of the colon of KO mice showed that the number of proliferating cells, as revealed by Ki-67 proliferation marker immunostaining, was significantly larger than control littermates. Specifically, proliferating cells in the KO mice were detected in the base of the crypt and up to 60% of the crypt column. In contrast, in control littermates the proliferating cells were fewer and present mostly at the base of the crypt. We hypothesize that pathways emanating from the CaR lead to proliferation inhibition of colon-derived epithelial cells by a mechanism that involves beta-catenin Ser-552 dephosphorylation and that the genetic ablation of this receptor promotes hyperproliferation of colonic crypts.